Chromatography is a method of separating complex chemical mixtures wherein individual chemical species of the mixture are distinguished from each other through selective distribution among heterogeneous phases for analytical and/or preparative purposes.
The distribution process is a dynamic process wherein the mixture is passed through a bed of particulate material and species of the mixture, having greater or lesser affinity to the bed material, are distinguished from others by being retained longer or passing through the bed more quickly. A mobile phase is used to carry the species through the column.
The mobile phase can comprise either a gas, liquid or supercritical fluid, thus a chromatographic method is referred to as gas chromatography, liquid chromatography or supercritical fluid chromatography. Separation of the mixture is based upon physical and/or chemical principles of adsorption and partition phenomena and thus the consistency of the bed and the materials comprising the bed control the attainment of repeatable accuracy and sensitivity for separation and/or identification of components.
The bed of a chromatographic apparatus comprises a column of particulate material, through which the mixture is percolated by gravity, electroosmotic flow or various pressures. In liquid chromatography, a contemporary direction is the utilization of high performance systems wherein the complex mixture, in a liquid mobile phase, is pumped at high pressure through a packed column of bed material.
In a typical preparative high performance liquid chromatographic system a mixture of compounds in solution is pumped at high pressure into a packed column of selected bed material. By means of the appropriate selection of bed material and liquid mobile phase, the column retains the components of the mixture to enable the discrete elution and separate collection of distinguished components of the mixture.
Generally, the size of the columns used in chromatography are dependent upon whether the system is gas or liquid and whether the use is for analytical or preparative purposes. Recent high performance liquid chromatographic systems have been developed which utilize analytical semi-preparative processes. Regardless of the size of the column, the overall performance of the column is highly dependent upon the manner by which the column is packed to form the bed and the consistency of the packing within the bed.
Generally, in preparing a packed column, a carefully proportioned slurry is formed comprising the bed material. The slurry is inserted into the column and liquid comprising the slurry is removed from the column to form a bed of particulate material in a packed arrangement. As the internal diameter of a column is decreased, particularly for use in capillary liquid chromatographic apparatus, processes for packing the column have become critical and tedious to enable a tight homogenous distribution of particulate material without voids or particle agglomeration, and comprising a consistent interspacial relationship between packed particles. Thus, there has been difficulties associated with the preparation of packed columns, particularly capillary columns which have an internal diameter of about 500 microns or less. The processes currently being used to pack capillaries are generally viewed as being tedious and having undesirable failure rates due to inconsistent homogeneity of the final packed capillary.
Most methods of packing currently used involve the application of axial compression forces at an end of the column bed to drive the bed material into the column with the subsequently entering material pushing axially against the first entering material to enable packing. One typical such method is a hydraulic packing system wherein a column is fritted at one end to enable passage of liquid but resist passage of particulate bed material. A defined liquid slurry of packing material is pushed into the opposite end of the column under high pressure or by a compression member which essentially constitutes a ram, sized to enter the interior diameter of the column and axially push slurry, trapped between it and the fritted opening of the column, into position within the column. Such elaborate ram driving apparatus, is generally only able to pack a single column at a time, requires significant apparatus modification for changes in column size and diameter and is subject to production of packed columns having significant failure rates for homogeneity of bed material.
Another method conventionally being used, embodies the application of radial compression on a flexible capillary wherein a pliant capillary, and thus the bed material, is radially squeezed by pneumatic or hydraulic pressure to achieve homogeneity. Systems using such method have not solved the problem of homogeneity failure and add the problem of testing the durability of the column.
Still another method which is currently used is an electrophoretic process wherein a voltage is imposed across a capillary, generating an electroosmotic flow that causes the particles to flow toward an attracting polarity and enable packing. Such process is inexpensive in comparison to axial or radial pressure systems but has not been found suitable for use with small particles, such as sub-micron particles which may comprise the bed of small diameter capillaries and is dependent upon the surface chemistry of the particles, thus limiting the materials with which it can be used.
An object of the present invention is to provide a simple and effective method for packing chromatography columns.
A further object of the invention is to provide an effective method to pack capillary columns having an internal diameter of about 500 microns and less.
Another object of the invention is to provide an apparatus for packing chromatographic columns which is convenient to use and has a low rate of packing failures.
A still further object of the invention is to provide packed capillary columns, having an internal diameter of less than about 500 microns, which have improved homogeneity of the packed bed along their axial length.
Still another object of the invention is to provide packed capillary columns, having an internal diameter of less than about 500 microns, which have improved homogeneity of the packed bed along its axial length.
These and other objects of the invention will be apparent from the following description of the invention.